CASEIN 37 



Only when a protein has been prepared in crystalline form or 

 by many different observers in many different ways and has 

 always been found to possess the same physical, physico-chemical 

 and chemical constants, can we pronounce it, with any degree 

 of safety, a definite chemical individual. So far this has only 

 been accomplished for a very limited number of proteins. 



It cannot with propriety be assumed, therefore, that the fol- 

 lowing methods for the isolation of "pure" proteins yield definite 

 chemical individuals, save those for the isolation of the crystal- 

 line proteins and of casein and of the various members of the 

 protamin group. But the methods which follow are those which, 

 to the knowledge of the author, have been shown to yield, at 

 least, mixtures of appreciably uniform composition, appreciably free 

 from non-protein contamination. 



Very many well-known methods for the isolation and purifi- 

 cation of various proteins are, of course, omitted, not because 

 they do not in all probability yield products conforming to the 

 above restrictions, but because either evidence of this fact is 

 lacking, or they have never been employed in research of a dis- 

 tinctly physico-chemical character.* 



2. Casein. The following is the method of preparation 

 employed by Hammarsten (8) : 



"Milk is diluted with four volumes of water and the mixture 

 treated with acetic acid to 0.75 to 1 per mille. Casein thus ob- 

 tained is purified by repeatedly dissolving in water with the aid 

 of the smallest quantity of alkali possible, by filtering and re- 

 precipitating with acetic acid and thoroughly washing with water. 

 Most of the milk-fat is retained by the filter on the first filtration, 

 and the casein contaminated with traces of fat is purified by 

 treating with alcohol and ether." 



According to Bosworth and Van Slyke (3) casein prepared in 

 this manner is always contaminated by a small admixture of 

 dicalcium phosphate and contains about 0.14 per cent of phos- 

 phorus attributable to this source. The method of preparation 

 which these investigators employ to secure a product free from 

 this contamination is as follows: 



Separator skim-milk is diluted with seven or eight times its 

 volume of distilled water, and dilute acetic acid (6 cc. of glacial 



* For a number of methods for the separation of various animal proteins 

 vide Hammarsten (8). 



